Structural and functional characterization of a metagenomically derived γ‐type carbonic anhydrase and its engineering into a hyperthermostable esterase

Abstract

<p>The 16S microbial community profiling of a metagenomics library from geothermal spring at Lisvori (Lesvos island, Greece) enabled the identification of a putative sequence exhibiting 95% identity to the γ-type carbonic anhydrase (γ-CA) from <em>Caloramator australicus</em> (γ-<em>Ca</em>CA). The sequence of γ-<em>Ca</em>CA was amplified by PCR, cloned, and expressed in <em>E. coli</em>. Activity assays showed that γ-<em>Ca</em>CA possesses very low, but detectable, anhydrase activity, while exhibiting no measurable esterase activity. Differential scanning fluorimetry (DSF) revealed that the enzyme shows high thermal stability with a melting temperature (<em>T</em>_<em>m</em>) approximately 65–75°C in the pH range between 5.5 and 9.0. The structure of γ<em>-Ca</em>CA was determined by X-ray crystallography at 1.11 Å resolution, the highest resolution reported so far for a γ<em>-</em>CA. The enzyme was crystallized as a trimer in the crystallographic asymmetric unit and contains three zinc-binding sites, one at each interface of neighboring subunits of the trimer. Structure-based rational design enabled the design and creation of a mutant enzyme (γ<em>-Ca</em>CAmut) which possessed a heptapeptide insertion at the active-site loop and two-point mutations. Kinetic analysis demonstrated that γ-<em>Ca</em>CAmut was successfully converted into a catalytically active esterase indicating successful activity gain through structure-guided engineering. The thermostability of γ-<em>Ca</em>CAmut was significantly increased, aligning with the thermostability typically observed in hyperthermostable enzymes. X-ray crystallographic analysis of the γ-<em>Ca</em>CAmut structure at 2.1 Å resolution, provided detailed structural insights into how the mutations impact the overall enzyme structure, function, and thermostability. These findings provide valuable structural and functional insights into γ-CAs and demonstrate a strategy for converting an inactive enzyme into a catalytically active form through rational design.<br></p>